Tumor antigen based on products of the tumor suppressor gene WT1

ABSTRACT

A tumor antigen that comprises, as an active ingredient, a product of the Wilms&#39; tumor suppressor gene WT1 or a peptide composed of 7-30 contiguous amino acids containing an anchor amino acid for binding to major histocompatibility complex (MHC) class I in said amino acid sequence, and a vaccine comprising said antigen.

CONTINUING APPLICATION INFORMATION

This application is a Continuation of U.S. application Ser. No.09/744,815, filed on Jan. 30, 2001 now U.S. Pat. No. 7,030,212, which isa National Stage of International Application Serial No. PCT/JP99/04130,filed on Jul. 30, 1999.

TECHNICAL FIELD

The present invention relates to tumor antigens based on the products ofWT1, the tumor suppressor gene of Wilms tumor. The tumor antigens areuseful as anti-cancer vaccines against tumors of the blood such asleukemia, myelodysplastic syndromes, multiple myeloma, and malignantlymphoma, or solid tumors such as gastric cancer, colon cancer, lungcancer, breast cancer, germ cell tumor, hepatic cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer, andovarian cancer, as well as all cancers that express WT1.

BACKGROUND ART

The immunological mechanisms to reject foreign material are generallycomprised of: the humoral immunity which involves macrophages thatrecognize antigens and function as antigen presenting cells, helper Tcells that activate other T cells etc. by recognizing antigen presentedby said macrophages and then producing various cytokines, B cells thatdifferentiates into antibody-producing cells by the action of saidlymphokines etc., as well as the cellular immunity in which killer Tcells that undergo differentiation in response to antigen presentation,and attack and destroy the target cells.

At present, cancer immunity is mainly considered to be derived fromcellular immunity in which killer T cells participate. In the killer Tcell-involved cancer immunity, precursor T cells that recognized tumorantigen presented in the form of a complex between the majorhistocompatibility complex (MHC) class I and the tumor antigendifferentiate and propagate to produce killer T cells, which attack anddestroy tumor cells. At this time, tumor cells present, on the surfacethereof, the complex of the MHC class I antigen and the tumor antigen,which is targeted by the killer T cells (Cur. Opin. Immunol., 5: 709,1993; Cur. Opin, Immunol, 5: 719, 1993; Cell, 82: 13, 1995; Immunol.Rev., 146: 167, 1995).

The above tumor antigen presented by the MHC class I antigen on thesurface of the target tumor cells is considered to be a peptide composedof about 8 to 12 amino acids produced after the antigen proteinsynthesized in the tumor cells underwent processing by intracellularproteases (Cur. Opin. Immunol., 5: 709, 1993; Cur. Opin, Immunol, 5:719, 1993; Cell, 82: 0.13, 1995; Immunol. Rev., 146: 167, 1995).

Currently, antigen proteins are being searched for various cancers, butfew have been demonstrated as cancer specific antigens.

WT1, a Wilms tumor suppressor gene (WT1 gene) was isolated fromchromosome 11p13 as one of the causative genes of Wilms tumor based onthe analysis of the WAGR syndrome that was complicated by Wilms tumor,aniridia, urogenital anomaly, mental retardation, etc. (Gessler, M. etal., Nature, 343: 774-778 (1990)), and the genomic DNA is about 50 K^(b)and is composed of ten exons, of which cDNA is about 3 kb. The aminoacid sequence deduced from the cDNA is as set forth in SEQ ID NO: 1(Mol. Cell. Biol., 11: 1707, 1991).

From the facts that the WT1 gene is highly expressed in human leukemiaand that the treatment of leukemia cells with WT1 antisense oligomersresults in suppression of cellular growth (Japanese Unexamined PatentPublication (Kokai) No. 9-104627), the WT1 gene has been suggested topromote the growth of leukemia cells. Furthermore, WT1 was found to behighly expressed in solid tumors such as gastric cancer, colon cancer,lung cancer, breast cancer, lung cell cancer, hepatic cancer, skincancer, bladder cancer, prostate cancer, uterine cancer, cervicalcancer, and ovarian cancer (Japanese Patent Application (Tokugan)9-191635), and the WT1 gene was demonstrated to be a new tumor marker inleukemia and solid tumors. However, it has not been confirmed that theexpression products of the WT1 gene are tumor-specific antigens usefulas a cancer vaccine.

DISCLOSURE OF THE INVENTION

Thus, the present invention intends to confirm the possibility that theexpression product of the WT1 gene is a tumor antigen and to provide anew tumor antigen.

After intensive research to resolve the above problems, the inventors ofthe present invention have synthesized polypeptides having 7 to 30contiguous amino acids containing at least one amino acid that isexpected to function as an anchor amino acid in the binding with mouseand human MHC class I and MHC class II in the amino acid sequence of theexpression product of the WT1 gene, confirmed that these peptides bindto MHC proteins and, when bound to the MHC class I antigen, inducekiller T cells and exert cytocidal effects on the target cell, andthereby have completed the present invention.

Thus, the present invention provides a tumor antigen comprising anexpression product of mouse WT1 or a portion thereof. According to apreferred embodiment, the present invention provides a tumor antigenthat comprises, as an active ingredient, a peptide having 6 to 30 aminoacids containing an anchor amino acid required for binding to the MHCmolecules in the amino acid sequence as set forth in SEQ ID NO: 1 thatcorresponds to the cDNA of WT1.

Furthermore, the present invention provides a tumor antigen thatcomprises, as an active ingredient, a peptide having 7 to 30 amino acidscontaining an anchor amino acid for binding to the MHC molecules in theamino acid sequence as set forth in SEQ ID NO: 2 that corresponds to thecDNA of human WT1.

The present invention also provides a cancer vaccine comprising theabove tumor antigen.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing a ratio of CD4⁺ and CD8⁺ cells in flowcytometry on the cells immunized with the D^(b) 126 peptide andnon-immunized cells in Example 1.

FIG. 2 is a graph that compares the cytocidal effect of the cellsimmunized with the D^(b) 126 peptide on the target cells pulsed withD^(b) 126 peptide and the non-pulsed target cells in Example 2.

FIG. 3 is a graph having the same meaning as in FIG. 2.

FIG. 4 is a graph in which A represents the cytocidal effect of CTLinduced by the D^(b) 126 peptide on the T2 cells pulsed with the D^(b)126 peptide in Example 3, and B represents the cytocidal effect of CTLinduced by the WH 187 peptide on the T2 cells pulsed with the WH 187peptide in Example 3.

FIG. 5 is a chart showing the result of FACS analysis on the surfacemarkers of CTL induced by the D^(b) 126 peptide (CD19 cells and CD3cells).

FIG. 6 is a similar chart to FIG. 5 with respect to the CD4 cells andthe CD8 cells.

FIG. 7 is a similar chart to FIG. 5 with respect to the CD56 cells.

FIG. 8 is a chart showing the result of FACS analysis on the surfacemarkers of CTL induced by the WH 187 peptide (CD19 cells and CD3 cells).

FIG. 9 is a chart similar to FIG. 8 with respect to the CD4 cells andthe CD8 cells.

FIG. 10 is a chart similar to FIG. 8 with respect to the CD56 cells.

FIG. 11 is a graph showing the effect of anti-HLA-A2.1 antibody on thespecific lysis of T2 cells pulsed with the D^(b) 126 peptide by theD^(b) 126 peptide-specific CTL.

FIG. 12 is a graph comparing the lytic activity of the D^(b) 126peptide-specific CTL on the target cells expressing or not expressingWT1. In the figure, a shows the result obtained when the E:T ratio is7.5:1 and b shows the result obtained when the E:T ratio is 15:1.

FIG. 13 is a graph that compares the lytic activity of the D^(b) 126peptide-specific CTL on the tumor cells (FBL3) that inherently expressWT1 and the tumor cells (RMA) that do not express WT1.

FIG. 14 is a graph that compares the lytic activity of the D^(b) 126peptide-specific CTL on the cells that were transformed with the WT1gene and the same cells that were not transformed.

FIG. 15 is a graph showing the effect of anti-H-2 D^(b) antibody on thecytotoxicity of the D^(b) 126 peptide-specific CTL.

FIG. 16 is a graph showing the in vivo immunological effect when micewas immunized with the D^(b) 126 peptide as a vaccine.

FIG. 17 is a graph showing the immunological effect when a plasmidexpressing WT1 is administered to mice as a DNA vaccine.

FIG. 18 is a graph showing the absence of the immunological effect whena plasmid not expressing WT1 is administered.

BEST MODE FOR CARRYING OUT THE INVENTION

In accordance with the present invention, K^(b) and D^(b) of mouse MHCclass I and A* 0201 of human HLA were selected as a basis for designingtumor antigen peptides, and peptides expected to have a high affinitywith them were selected.

Based on the description in Immunogenetics 41: 178-228 (1995), Phe andTyr at position 5 and Leu and Met at position 8 etc. are expected to beanchor amino acids for binding to K^(b), and Asn at position 5 and Metand Ile at position 9 etc. are expected to be anchor amino acids forbinding to D^(b).

It is also known that the size of the tumor antigen peptide presented byMHC class I on the surface of tumor cells is about 8 to 12. Therefore,the tumor antigen peptide of the present invention is a peptide having 7to 30 contiguous amino acids containing an anchor amino acid in theamino acid sequence of the WT1 gene expression product as set forth inSEQ ID NO: 1. The number of amino acids is preferably 8 to 12, forexample 8 or 9.

As a specific embodiment in the present invention, the followingpeptides comprising 8 amino acids: K^(b) 45 Gly Ala Ser Ala Tyr Gly SerLeu (SEQ ID NO: 3), and

K^(b) 330 Cys Asn Lys Arg Tyr Phe Lys Leu (SEQ ID NO: 4) were used aspeptides binding to K^(b) of MHC class I, and the following peptidescomprising 9 amino acids:

D^(b) 126 Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 5),

D^(b) 221 Tyr Ser Ser Asp Asn Leu Tyr Gln Met (SEQ ID NO: 6), and

D^(b) 235 Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO: 7)

were used as peptides binding to D^(b) of MHC class I. The underlinedamino acids in the above sequences are those amino acids that areexpected to function as anchors.

Then, among these peptides, for K^(b) 45 and K^(b) 330 the bindingactivity with K^(b) of MHC class I was measured, and for D^(b) 126,D^(b) 221 and D^(b) 235 the binding activity with D^(b) of MHC class Iwas measured using the cell line (RMA-S) that does not express theendogenous antigen peptide (empty) and the cell line (RMA-S) thatexpresses K^(b) and D^(b) molecules.

Thus, RMA-S was cultured at 26° C. to effect high expression of MHCclass I, and the cultured cells were incubated with the solutions of thetest peptides at 37° C. for 1 hour. This makes unstable the MHC moleculethat does not bind to the peptide leading to their disappearance fromthe cell surface and leaving MHC class I molecules alone that bound tothe peptide. Then using fluorescently labeled monoclonal antibody thatrecognizes MHC class I (K^(b), D^(b)), RMA-S cells were stained.Finally, the binding dissociation constant was calculated from theaverage amount of fluorescence per cell (Immunol. Lett., 47: 1, 1995).

As a result, the following result was obtained:

K^(b) 45 −4.5784838 (log)

K^(b) 330 −5.7617732

D^(b) 126 −6.2834968

D^(b) 221 −5.7545398

D^(b) 235 −6.1457624

As hereinabove stated, both have a strong to moderate binding affinity(Kd value) with K^(b) or D^(b), and the D^(b) 126 peptide having thehighest binding affinity was used in the following experiment.

For humans also, based on the description in Immunogenetics 41: 178-228(1995), Leu and Met at position 2 from the N-terminal and Val and Leu atposition 9 from the N-terminal are expected to be anchor amino acids forbinding to HLA-A* 0201. Thus, the following two peptides having 9 aminoacids that meet the above requirement were synthesized in the amino acidsequence of human WT1 protein (Mol. Coll. Biol., 11: 1707-1712, 1991)(SEQ ID NO: 2):

D^(b) 126; Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 5)

(the same as the sequence Of D^(b) 126 in mouse)

WH 187; Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 8)

(anchor amino acids are underlined).

The binding activity of the above peptides with HLA-A* 0201 was measuredas follows:

The above peptides and T2 cells having the empty HLA-A* 0201 molecules(J. Immunol., 150: 1763, 1993; Blood, 88: 2450, 1996) were incubated at37° C. for 1 hour, and then the T2 cells were stained with fluorescentlylabeled monoclonal antibody that recognizes HLA-A2.1 to calculate thebinding dissociation constant based on the average amount offluorescence per cell in the FACS analysis.

Binding activity Peptide Kd (M) D^(b) 126 1.89 × 10⁻⁶ WH 187 7.61 × 10⁻⁶

The two peptides have a binding affinity of moderate degree or higher.

Using the above D^(b) 126 and WH 187 as a peptide that can combine withhuman MHC Class I molecules, the experiment described hereinafter wasperformed.

The present invention also relates to a cancer vaccine comprising theabove antigen as an active ingredient. This vaccine can be used forprophylaxis or treatment of tumors of the blood such as leukemia,myelodysplastic syndromes, multiple myeloma, and malignant lymphoma, orsolid tumors such as gastric cancer, colon cancer, lung cancer, breastcancer, germ cell tumor, hepatic cancer, skin cancer, bladder cancer,prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.The vaccine can be given via oral or parenteral administration such asintraperitoneal, subcutaneous, intradermal, intramuscular, intravenous,and nasal administration.

As a method of administering the vaccine of the present invention, therecan be used a method, in which mononuclear cells are collected from thepatient's peripheral blood, from which dendritic cells are removed, andthe peptide of the present invention is pulsed thereto, which is thenreturned to the patient via subcutaneous administration etc.

Vaccines can contain, in addition to the peptide given as the aboveactive ingredient, pharmaceutically acceptable carriers for examplesuitable adjuvants such as a mineral gel like aluminum hydroxide; asurfactant such as lysolecithin, pluronic polyol; a polyanions; apeptide; or an oil emulsion. Alternatively, other aggregates that can bemixed into liposomes or blended into polysaccharides and/or vaccines canbe used. The dosage is generally 0.1 μg to 1 mg/kg per day.

In the present invention, DNA encoding the above polypeptide vaccine canalso be used as a vaccine (DNA vaccine). Thus, after a nucleic acidencoding WT1 or a portion thereof, preferably DNA, is inserted to asuitable vector, preferably an expression vector, it is administered toan animal to produce cancer immunity. A specific example is shown inExample 9.

EXAMPLES

Then, the following examples will demonstrate that the peptide of thepresent invention is useful as a tumor antigen or a cancer vaccine.

Example 1

One hundred μg of the D^(b) 126 peptide, 200 μg of porcine lactatedehydrogenase and 0.5 ml of Freund's incomplete adjuvant wereintraperitoneally injected to C57BL/6 mice twice every week forimmunization treatment. One week after the immunization treatment, themouse spleen was extracted, from which suspensions of spleen cells wereprepared. On the other hand, the irradiated spleen cells of thesyngeneic mice pulsed with the D^(b) 126 peptide were incubated with asolution containing 50 μg/ml peptide at 37° C. for 30 minutes, which wasused as the antigen presenting cell.

The above immunized spleen cells and the irradiated spleen cells wereco-cultured for 5 days to induce or prepare killer T cells. On the otherhand, using the Europium labeled EL-4 cells (expressing K^(b) and D^(b))pulsed with the D^(b) 126 peptide (incubated at 37° C. for 30 minuteswith a 100 μg/ml peptide solution) as the target cell in a standardmethod, a killing assay was performed in the following procedure (Table1).

As a result, when the EL-4 cells pulsed with D^(b) 126 were used as thetarget, cytocidal effects were observed, but when the EL-4 cells notpulsed with D^(b) 126 were used as the target, few cytocidal effectswere observed.

TABLE 1 Mouse A Mouse B Peptide + 76.6% 37.2% Peptide − 4.9% 0.9% E/Tratio 40:1

Then, the spleen samples that exhibited significant cytocidal effects inthe killing assay were stained with the fluorescently labeled anti-CD4antibody or anti-CD8 antibody, which were then subjected to flowcytometry to analyze the expression of CD4 and CD8.

As a result, as shown in FIG. 1, in the spleen cells immunized with theD^(b) 126 peptide, there was an increase in the CD8⁺ cells representedby the killer T cells and the ratio of the CD8⁺ cells to the CD4⁺ cellsrepresented by the helper T cells etc. was inversely increased comparedto the non-immunized control spleen cells.

Example 2

Dendritic cells (DC) derived from the bone marrow of the C57BL/6 micewere prepared in the following manner. According to the standard method,the bone marrow cells were cultured in the presence of GM-CSF to preparebone marrow-derived dendritic cells (J. Exp. Med. 182: 255, 1995).

The dendritic cells cultured for 7 days, 10 μM OVAII (Ovalbumin II) and1 μM D^(b) 126 peptide were incubated for 3 hours and then washed.

Then, the above DC cells were intradermally injected to the foot padsand hands of C57BL/6 mice, and on day 5 the lymph nodes were removed toprepare cell suspensions. On the other hand, the B7.1-RMA-S cells (RMA-Scells transfected with a gene encoding B7.1 which is a co-stimulatorymolecule) pulsed with D^(b) 126 and irradiated were prepared.

Then the above cell suspension derived from the relevant lymph node andthe B7.1-RMA-S cells were mixed and cultured for in vitro restimulation.

Then, on day 5 after the in vitro restimulation, a killing assay wasperformed using the ⁵¹Cr-labeled RMA-S cells as the target. When ⅛ ofthe total lymphocytes recovered on day 5 after restimulation was used asthe effector cell, the E/T ratio was set as the highest one (1.0).

As shown in FIGS. 2 and 3, the effector cells derived from the lymphnodes of the mice immunized with the D^(b) 126 peptide killed the targetcells pulsed with said peptide but did not kill the target cells thatwere not pulsed with said peptide.

Analysis of the ratio of the CD4⁺ cells and the CD8⁺ cells by flowcytometry performed as in Example 1 shows that CD4: CD8=1:1.4 to 1.7 andthat in the mouse cells immunized with the D^(b) 126 peptide the CD8⁺cells were increased and the ratio of the CD4⁺ cells:the CD8⁺ cells(about 2:1 in the control cells) was reversed in the cells immunizedwith the D^(b) 126 peptide as compared to the non-immunized mouse(control) cells.

Example 3

T2 cells (5×10⁴) that were irradiated after incubating for 1 hour withthe peptide D^(b) 126 or WH 187 (40 μg/ml) and the peripheral bloodmononuclear cells (1×10⁶) from a healthy human having HLA-A* 0201 wereco-cultured. One week later, T2 cells that were irradiated afterincubating for 1 hour with the peptide (20 μg/ml) were added to theabove culture system for restimulation. From the following day, humanIL-2 (final concentration 100 JRU/ml) was added to the culture.

Subsequently, after repeating, for five times, stimulation with the T2cells that were irradiated after being pulsed with the peptide, akilling assay was performed using, as the target, the T2 cells pulsedwith the peptide or the T2 cells not pulsed with the peptide. Thesurface markers of the induced CTL were subjected to FACS analysis.

The killing assay was performed according to the standard method using,as the target, the Europium-labeled T2 cells pulsed with the peptide.

Effector: Target ratio (E/T ratio) is 10:1

Co-cultivation time: 3 hours

The concentration of the peptide in the culture 5 μg/ml

The result is shown in FIG. 4. A in FIG. 4 shows the cytocidal effect ofCTL induced using D^(b) 126 peptide on the T2 cells pulsed with theD^(b) 126 peptide, and B in FIG. 4 shows the cytocidal effect of CTLinduced using the WH 187 peptide on the T2 cells pulsed with the WH 187peptide.

In either case, more potent cytocidal effects were observed in the T2cells pulsed with the peptide.

The results of FACS analysis are shown in FIGS. 5 to 10. FIGS. 5 to 7show the results of human CTL induced with the D^(b) 126 peptide,indicating that most of the cells were CD8-positive. FIGS. 8 to 10 showthe results of human CTL induced with the WH 187 peptide. TheCD4-positive cells and the CD8-positive cells were almost equal in thenumber.

Example 4

In order to test the MHC dependency of the cytolytic activity of theD^(b) 126 peptide-specific CTL, anti-HLA-A2.1 monoclonal antibody wasused to block the cytolytic activity of CTL on the T2 cells pulsed withthe peptide. The specific cytolysis of the T2 cells pulsed with theD^(b) 126 peptide was measured in the presence or absence of monoclonalantibody (BB7.2) that blocks HLA-A2.1 molecule at a E/T ratio of 5:1.

The result is shown in FIG. 11. In the figure, the * symbol representsthe result obtained using anti-H-2 K^(b) monoclonal antibody in stead ofanti-HLA-A2.1 monoclonal antibody. As can be seen from the figure, theaddition of 60 μg/ml of anti-HLA-A2.1 monoclonal antibody resulted inthe reduction of the cytotoxicity to background of the cytolysis of theT2 cells. Unrelated monoclonal antibody (anti-H-2 K^(b) monoclonalantibody Y3) with the same isotype had no effects on the lysis of the T2cells.

Example 5

It was tested whether the D^(b) 126 peptide-specific CTL can kill theHLA-A2.1-positive leukemia cells that inherently express WT1. As thetarget cell, the TF1 cells (express WT-1, HLA-A2.1-positive), the JYcells (do not express WT-1, HLA-A2.1-positive), and the Molt-4 cells(express WT1, HLA-A2.1-negative) were used and cytotoxicity was measuredat a E:T ratio of 7.5:1 (a) or 15:1 (b).

The result is shown in FIG. 12. The D^(b) 126 peptide-specific CTLexhibited a significant cytotoxicity to the HLA-A2.1-positive leukemiacell TF1 that inherently expresses WT1, but exhibited a cytolysis of abackground level to the Molt-4 (which expresses WT1, HLA-A2.1-negative)or the JY cells (which do not express WT1, HLA-A2.1-positive).

Example 6

It was tested whether the D^(b) 126 peptide-specific CTL can recognizetumor cells that inherently express WT1 and can cause cytolysis thereof.Specific lysis was measured at the E/T ratio shown in FIGS. 13 and 14for tumor cells (FLB3) that express WT1 and tumor cells (RMA) that donot express WT1 (FIG. 13) or for the C1498 cells transfected with theWT1 gene or the C1498 cells not transfected with the WT1 gene (FIG. 14).

As shown in FIG. 13, the D^(b) 126 peptide-specific CTL caused lysis ofthe FBL3 cells that inherently express WT1 but not the RMA cells that donot express WT1. As shown in FIG. 14, the D^(b) 126 peptide-specific CTLfurther killed the C1498 cells transfected with the mouse WT1 gene ascompared to the parent C1498 cells that do not express WT1. Thisconfirmed that the molecule targeted for cell killing by CTL is indeedthe WT1 peptide. These results suggest that the D^(b) 126peptide-specific CTL can recognize D^(b) 126 peptide or the relatedpeptides, which were naturally produced by the intracellular processingof the WT1 protein and presented on the H-2 D^(b) molecules of theWT1-expressing cells.

Example 7

In order to test whether the cytolytic activity of CTL is. MHCdependent, measurement was performed in the presence of an antibodyagainst the H-2 class I molecule. Thus, cytolytic activity of the D^(b)126 peptide-specific CTL against the RMA-S cells pulsed with the D^(b)126 peptide was measured in the presence of a titer-adjusted monoclonalantibody against H-2 K^(b) (28.13.3S), H-2 D^(b) (28.11.5S), or H-2Ld(MA143). As the control monoclonal antibody, monoclonal antibody havingthe same isotype was used.

The result is shown in FIG. 15. Depending on the increasedconcentrations of antibody against H-2 D^(b), the lytic activity of CTLagainst the RMA-S cells pulsed with the D^(b) 126 peptide wassuppressed, whereas antibodies against H-2 K^(b) or H-2Ld did notsuppress the lytic activity of CTL. These results indicate that CTLexhibits the cytolysis activity in a H-2 D^(b)-dependent manner.

Example 8

It was tested whether in vivo tumor immunity can be elicited by theactive immunization with the D^(b) 126 peptide. Using the LPS-activatedspleen cells (solid line in FIG. 16) pulsed with the D^(b) 126 peptide,the LPS-activated spleen cells only (shaded line) or phosphate bufferedsaline (PBS) only (broken line), mice were immunized once every week.After immunization for 3 weeks, 3×10⁷ FBL3 leukemia cells wereintraperitoneally administered.

The result is shown in FIG. 16. The mice immunized with the D^(b) 126peptide overcame tumor challenge and survived, whereas the non-immunizedmice and the mice immunized with the LPS-activated spleen cells couldnot reject tumor challenge and died. In both of the immunized andnon-immunized mice, the presence of ascites was observed three daysafter the above intraperitoneal injection of tumor cells. Ascitescontinued to increase in the non-immunized mice, and the mice eventuallydied. In the immunized mice, on the other hand, ascites started togradually decrease thereafter, and the mice completely rejected tumorchallenge and survived. In the non-immunized mice, natural regressionwas occasionally observed. The regression is expected to be due tonatural induction of CTL specific for Friend leukemia virus (FBL3leukemia cells are transformed with this virus). Because such CTLinduction has occasionally been observed in C57BL/6 mice.

Example 9 DNA Vaccine

One hundred μg of WT1-expressing plasmid DNA (plasmid that continuouslyexpresses WT1 which was prepared by ligating the Sau 3AI fragment ofmouse WT1 cDNA (Molecular and Cellular Biology, vol. 11, No. 3, p.1707-1712 (1991), the left column on p. 1709) to the CMV-IE promoter)(Proc. Natl. Acad. Sci. USA., 92: 11105-11109 (1995)) wasintramuscularly injected to 6 to 8 week old C57BL/6 mice every 10 daysfor a total of three times. Ten days after the last intramuscularinjection, mouse spleens were removed to prepare spleen cells. After thespleen cells and mWTlC1498 cells (irradiated with 40 Gy radiation)expressing WT1 were co-cultured at 37° C. for 6 days, a killing assay(Europium-labeled) was performed using C1498 (PM5G-mWT1) that expressedWT1 and C1498 (PM5G) that did not express WT1 as the target cell. Asused herein, C1498 is a mouse myelogenic leukemia cell line that doesnot express WT1.

Cytotoxic T cells (CTL) that kill C1498 (PM5G-mWTl) cells that areexpressing WT1 but do not kill C1498 cells (PM5G) that are notexpressing WT1 were induced.

The result is shown in FIG. 17.

As a control, a similar experiment as the above was performed in whichplasmid that does not express WT1 (contain no WT1 CDNA) wasintramuscularly injected to mice in stead of plasmid that expresses WT1.As in the above experiment, spleen cells were removed. After in vitrostimulation with C1498 (PM5G-mWT1) cells that express WT1, a killingassay was performed.

As shown in FIG. 18, no WT1-specific CTL was induced by intramuscularinjection of the control plasmid DNA having no WT1 CDNA.

The above results demonstrated that the peptide of the present inventionindeed functions as a tumor antigen and that it induced the growth ofkiller T cells (tumor cell-toxic T cells) against tumor cells.Therefore, the tumor antigen peptide of the present invention is usefulas a cancer vaccine for leukemia and solid tumors that are accompaniedby increased expression of the WT1 gene.

1. An isolated polypeptide which consists of 7 to 30 contiguous aminoacid residues of WT1 protein, wherein the polypeptide binds to MHCproteins, and the polypeptide induces killer T cells when bound to MHCclass I antigen.
 2. The polypeptide of claim 1, wherein the WT1 proteinis human WT1 protein.
 3. The polypeptide of claim 1, which consists of 8to 12 contiguous amino acid residues of WT1 protein.
 4. The polypeptideof claim 1, which consists of 8 contiguous amino acid residues of WT1protein.
 5. The polypeptide of claim 1, which consists of 9 contiguousamino acid residues of WT1 protein.
 6. The polypeptide of claim 1, whichconsists of 9 contiguous amino acid residues of SEQ ID NO: 2, whereinthe amino acid residue at position 2 of the contiguous amino acidresidues is leucine or methionine and the amino acid residue at position9 of the contiguous amino acid residues is valine or leucine.
 7. Thepolypeptide of claim 1, which binds to MHC class I antigen or MHC classII antigen.
 8. A composition suitable for pharmaceutical use, comprisingthe polypeptide of claim 1 and a pharmaceutically acceptable carrier. 9.The composition of claim 8, wherein the pharmaceutically acceptablecarrier is an adjuvant, surfactant, polyanion, peptide or oil.